Host
serine proteases are essential for the influenza virus life cycle because the viral haemagglutinin is synthesized as a precursor which requires proteolytic maturation. Therefore, we studied the activity and expression of
serine proteases in lungs from mice infected with
influenza and evaluated the effect of
serine protease inhibitors on virus replication both in cell culture and in infected mice.
RESULTS: Two different inbred mouse strains were investigated: DBA/2J as a highly susceptible and C57Bl/6J as a more resistant strain to influenza virus
infection. The
serine proteases from lung homogenates of mice exhibited pH optima of 10.00. Using the substrate Bz-Val-Gly-Arg-p-nitroanilide or in zymograms, the intensities of proteolysis increased in homogenates from both mouse strains with time post
infection (p.i.) with the mouse-adapted influenza virus A/Puerto Rico/8/34 (H1N1; PR8). In zymograms at day 7 p.i., proteolytic bands were stronger and numerous in lung homogenates from DBA/2J than C57Bl/6J mice. Real-time PCR results confirmed differential expression of several lung
proteases before and after infecting mice with the H1N1 virus. The most strongly up-regulated
proteases were Gzma, Tmprss4, Elane, Ctrl, Gzmc and Gzmb. Pretreatment of mouse and human lung cell lines with the
serine protease inhibitors AEBSF or pAB or a cocktail of both prior to
infection with the H1N1 or the A/Seal/Massachusetts/1/80 (H7N7; SC35M) virus resulted in a decrease in virus replication. Pretreatment of C57Bl/6J mice with either
AEBSF or a cocktail of
AEBSF and pAB prior to
infection with the H1N1 virus significantly reduced
weight loss and led to a faster recovery of treated versus untreated mice while pAB alone exerted a very poor effect. After
infection with the H7N7 virus, the most significant reduction of
weight loss was obtained upon pretreatment with either the
protease inhibitor cocktail or pAB. Furthermore, pretreatment of C57BL/6J mice with
AEBSF prior to
infection resulted in a significant reduction in the levels of both the H1N1 and H7N7
nucleoproteins in mice lungs and also a significant reduction in the levels of the HA transcript in the lungs of the H1N1--but not the H7N7-infected mice.
CONCLUSION: